A number of medical diagnostic procedures, including PET and SPECT utilize radiolabeled compounds. PET and SPECT are very sensitive techniques and require small quantities of radiolabeled compounds, called tracers. The labeled compounds are transported, accumulated and converted in vivo in exactly the same way as the corresponding non-radioactively labeled compound. Tracers or probes can be radiolabeled with a radionuclide useful for PET imaging, such as 11C, 13N, 15O, 18F, 61Cu, 62Cu, 64Cu, 67Cu, 68Ga, 124I, 125I and 131I, or with a radionuclide useful for SPECT imaging, such as 99Tc, 75Br, 61Cu, 153Gd, 125I, 131I and 32P.
PET creates images based on the distribution of molecular imaging tracers carrying positron-emitting isotopes in the tissue of the patient. The PET method has the potential to detect malfunction on a cellular level in the investigated tissues or organs. PET has been used in clinical oncology, such as for the imaging of tumors and metastases, and has been used for diagnosis of certain brain diseases, as well as mapping brain and heart function. Similarly, SPECT can be used to complement any gamma imaging study, where a true 3D representation can be helpful, for example, imaging tumor, infection (leukocyte), thyroid or bones.
Angiogenesis plays a vital role in tumor growth and metastatic spread. Tumor angiogenesis is a multi-step process characterized by chemotactic and mitogenic response of endothelial cells to angiogenic growth factors, proteolytic degradation of extracellular matrix, and modulation of endothelial cell interaction with extracellular matrix mediated by integrin receptors. Each of these steps may represent a potential target for the development of tumor angiogenic and metastatic diagnostics.
Integrins are a family of membrane-spanning adhesion receptors composed of noncovalently linked α and β subunits, which combine to form a variety of heterodimers with different ligand recognition properties. Several integrins have been shown to interact with polypeptide domains containing the Arg-Gly-Asp (“RGD”) amino acid sequence present in various extracellular matrix-associated adhesive glycoproteins. Besides cell adhesion to extracellular matrix, integrins also mediate intracellular events that control cell migration, proliferation and survival.
One member of the integrin family, αvβ3 integrin, plays a key role in angiogenesis. It interacts with several extracellular matrix proteins, such as vitronectin, fibrinogen, fibronectin, thrombin and thrombospondin, and cooperates with molecules such as metalloproteases, growth factors, and their receptors. Due to its numerous functions and relatively limited cellular distribution, αvβ3 integrin represents an attractive target for diagnostic and therapeutic intervention. In addition, findings that several extracellular matrix proteins, such as vitronectin, fibrinogen, and thrombospondin interact with integrins via the RGD sequence have lead to the development of synthetic linear and cyclic peptides containing RGD sequence for integrin targeting. See for example, DE 197 25 368, U.S. Pat. No. 5,849,692, U.S. Pat. No. 6,169,072, U.S. Pat. No. 6,566,491, U.S. Pat. No. 6,610,826 and WO 2005/111064.
Researchers have demonstrated in a number of human xenograft tumor models in mice that radiolabeled peptides containing the RGD motif can be used for non-invasive investigation of αvβ3 integrin expression. The development of non-invasive methods to visualize and quantify integrin αvβ3 expression in vivo complements the use of antiangiogenic therapy based on integrin antagonism. For example, non-invasive integrin imaging is first used to evaluate the efficacy of anti-integrin based therapeutics and, secondly, may be used as a tool for optimizing both favorable tumor targeting and in vivo kinetic properties of new drug candidates. Imaging can also be used to provide an optimal dosage regimen and time course for patient treatment based on receptor occupancy studies. Precise documentation of integrin receptor levels may allow for a more accurate selection of patients who will most likely benefit from anti-integrin based treatments.
Kessler and co-workers [1] developed the pentapeptide cyclo(-Arg-Gly-Asp-D-Phe-Val-) (“c(RGDfV)”) which showed both high affinity and selectivity for integrin αvβ3. To date, most integrin αvβ3 targeted PET studies have utilized the radiolabeling of c(RGDfV)-based antagonists due to their high binding affinities which range from nanomolar to subnanomolar range for monomeric and multimeric c(RGDfV) respectively. In the late 1990's, Haubner et al. [2] prepared a monomeric peptide c(RGDyV) labeled with 125I. This tracer possessed receptor-specific tumor uptake in vivo, however, the labeled peptide had rapid tumor washout and unfavorable hepatobiliary excretion, due to its high lipophilicity, thus limiting its imaging applications. In an effort to develop an imaging agent with more favorable properties, glycosylation of the lysine side chain of an RGD peptide analog, c(RGDyK), decreased both the tracer's lipophilicity and hepatic uptake [3]. The resultant F-18 labeled glycopeptide was then synthesized and imaged:

[18F]Galacto-RGD exhibits integrin αvβ3-specific tumor uptake in integrin-positive M21 melanoma xenograft mouse models (4-6, see also 19). When [18F]galacto-RGD was imaged in mice with an integrin negative cell line, the A431 human squamous cell carcinoma model, the tracer did not localize on tumor cells, but rather localized at tumor vasculaturization sites having integrin αvβ3 expression. Initial clinical trials results from both healthy volunteers and cancer patients showed that the tracer was both safe and effective in detecting certain lesions that were integrin-positive with reasonable contrast.
[18F]Galacto-RGD currently represents one promising integrin marker for PET imaging of angiogenesis. As a monomeric RGD peptide tracer, it has a relatively low tumor targeting efficacy. In addition, its clinical utility is severely limited because of its relatively low integrin binding affinity, modest tumor standard uptake values, and unfavorable pharmacokinetic behavior. Therefore, tumors with low integrin expression levels may not be detectable. In addition, prominent tracer accumulation in the liver, kidneys, spleen, and intestines was observed in both preclinical models and human studies resulting in difficult visualization of abdomen lesions. To add to its imaging drawbacks, the synthetic preparation of the tracer is labor intensive, time consuming and inefficient, thereby limiting its widespread availability to clinicians.
Conjugation of PEG (poly(ethyleneglycol)) (“PEGylation”) has been shown to improve many properties of peptides and proteins, including plasma stability, immunogenicity, and pharmacokinetics. Chen et al. [7-9] conjugated RGD-containing peptides with PEG moieties of different sizes and en route to preparing radioiodinated, 18F- and 64Cu-labeled derivatives. Attachment of the PEG group favorably affected the pharmacokinetics, tumor uptake and retention of the tracer in human xenograft mouse models. The biological uptake and distribution appears to depend strongly on the nature and quantity of the cyclic peptide as well as the size of the PEG moiety. In an effort to further improve the imaging of cyclic peptides by improving PK, tumor uptake and retention, two strategies focused on the incorporation of hydrophilic amino acids and multimerisation of RGD.
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